New cytotoxic compounds from the leaves of Caesalpinia benthamiana (Baill.) Herend. & Zarucchi (Fabaceae)

Objective: The incidence of multi-drug resistant cancer and the adverse effects associated with available chemotherapy have necessitated the search for new drug candidates. This study investigates the cytotoxic activity of Caesalpinia benthamiana. Materials and Methods: Column chromatography (CC) and preparative thin layer chromatography (PTLC) were used to isolate compounds. Structural elucidation was done by spectroscopic analysis. MTT assay was used to evaluate cytotoxicity of the compounds against three human adenocarcinoma cells, using methotrexate and dimethyl sulfoxide (DMSO) as positive and negative controls, respectively. CyQuant direct cell proliferation and caspase-3/7 green detection assays were used to investigate the dichloromethane fraction. IC50 values of isolated compounds were determined from sigmoidal dose-response curve. Results: Four new cytotoxic compounds, benthamianoate (2), benthamiacone (3), benthamianin (5) and benthamianol (6), and two known compounds, methyl gallate (1) and 2-methoxyacrylic acid (4) were identified. All the compounds were active with the new monoterpenoid characterized as benthamiacone exhibiting the highest activity (IC50 13.23-21.97 μg/ml) across cancer cell lines investigated. CyQuant direct cell proliferation assay showed significant reduction in the number of live carcinoma cells, while caspase-3/7 green detection assay showed significant increase in the number of dead carcinoma cells. Conclusion: This study revealed potential cytotoxic compounds which are here reported for the first time from C. benthamiana.


Introduction
Cancer, a disease characterized by uncontrolled cell division resulting from prolonged unrepaired DNA damage by electrophilic species, can arise in any part of the body (WHO, 2018). Cancer is a main cause of death, with approximately 9.6 million cancer-related deaths and 18.1 million new cancer cases in 2018 (Bray et al., 2018). It is responsible for 1 out of 6 deaths recorded for all diseases annually (Hannah and Max, 2018) with the developing nations of the world accounting for approximately 56% of new cases and 70% of cancer mortality worldwide (Bray et al., 2018).
In 2018, cancers of the lung, breast, colorectal, prostate, skin and stomach were the most commonly diagnosed ones worldwide, while the commonest mortality was due to cancers of tracheal, lung, colorectal, colon, stomach, liver and breast (WHO, 2018). Environmental factors such as physical, chemical, biological, and dietary carcinogens have been indicated as major causes of cancer disease (WHO, 2018).
The genus Caesalpinia, is probably the smallest genera of the family Fabaceae, with about 200 species. This genus has been used in folkloric healthcare against numerous diseases and various investigators have validated their folklore uses (Zamble et al., 2008). C. benthamiana is widely distributed in West and Central Africa and is usually found in humid, rain forest, waste ground localities, deciduous woodland and savanna (Osho, 2014). In Africa traditional medicine, it has been used in treating erectile dysfunction, toothache and venereal diseases (Zamble et al., 2008), urethral discharge, snakebites, cataract and other eye problems (Dickson et al., 2006), dysentery (Irvine, 1961), skin diseases and wounds (Dickson et al., 2012). In addition, previous studies documented its antiinflammatory (Moronkola et al., 2009), antidiarrheal (Mbagwu and Adeyemi, 2008), antioxidant, antimicrobial (Dickson et al., 2006), antifertility and vasorelaxing activities (Zamble et al., 2008).

Chemicals
Solvents were purchased from Sigma-Aldrich, Germany. Cell culture materials were from Invitrogen, (Carlsbad, CA, USA).
Methotrexate, penicillinstreptomycin suspension, trypan blue and the MTT salt were from Thermo Fisher Scientific (England).

General procedures
Silica gel (80-200 mesh, Merck; Germany) was used for column chromatography (CC). Silica gel 60 F254 plates (10 x 5 cm, 0.5 mm; Merck, Germany) were used for thin layer chromatography (TLC). Silica gel 60 F254 plates (20 x 20 cm, 0.5 mm; Merck, Germany) were used for PTLC. PerkinElmer Fourier transform infrared spectroscopy (FTIR). Spectrum BX spectrometer was used for infra-red (IR) experiments. Mass data were obtained from the atmospheric pressure chemical ionization mass spectroscopy (APCI-MS). NMR spectra were recorded in CD3OD on Bruker Ascend TM Spectrometer at 500 MHz ( 1 H NMR) and 150 MHz ( 13 C NMR). Distortionless Enhancement by Polarization Transfer (DEPT) experiments were used to differentiate different carbon atoms. 1 H homonuclear connectivity was identified by Correlation spectroscopy (COSY). Heteronuclear single quantum correlation (HSQC) helped to determine 1 H-13 C bound connectivity and HMBC used to determine two and three 1 H-13 C and 13 C-13 C bound connectivity. The MTT assay absorbance was recorded on Tecan F50 microplate reader (Thermo Fisher Scientific, England).

Plant material
Caesalpinia benthamiana fresh leaves were collected at Ode Ekiti, Nigeria, in July 2015. Identification and authentication were done at Forest Herbarium Ibadan (FHI) where a voucher specimen (FHI 110847) was deposited. The leaves were air dried for two weeks under shade at room temperature (20-25ºC). The dried leaves were powdered and stored in air-tight container prior to use.

Cell culture
Cell lines of prostate (PC3), breast (MCF-7) and lung (A549) were purchased from European Collection of Authenticated Cell Cultures (ECACC), Public Health, England. They were cultured in T75 flasks containing Dulbecco's modified eagle medium (DMEM) supplemented with 10% Fetal bovine serum (FBS) and 1% penicillin-streptomycin suspension. They were maintained in a humidified atmosphere, containing 5% CO2 at 37°C and changes in their morphology were checked using an inverted microscope (Evos Life Technology, UK) at magnifications X40 and X100 every 48 hr. Cells were sub-cultured and split in a 1:3 ratio into another flask when they reached confluence by washing with 0.02% Ethylene diamine tetra acetic acid (EDTA) in Phosphate-buffered saline (PBS)PBS and they were dissociated using 0.2% trypsin. A manual haemocytometer (Hausser Scientific Company, New York USA) was used to count the cells.

Fractions and compounds preparation
Fractions and pure compounds were dissolved in DMSO to give 200 μg/ml stock solution and serial dilutions were prepared to produce 100 and 50 µg/ml (fractions) and 100, 50, 25 and 12.5 µg/ml (compounds). Methotrexate was used at 100, 50, 25 and 12.5 µg/ml. Final concentration of DMSO in each sample was less than 0.1% (Basar et al, 2015).

In vitro MTT assay
Cytotoxicity was evaluated using MTT assay (Deepa et al., 2011). Cells were seeded into 24-well plates and incubated at 37°C in humidified CO2 (5%) for 24 hr after which the old medium was replaced with serum-free FBS. Then, 0.2 μl of test compounds in triplicates was added onto the well containing cells except the blank well. The plates were again incubated for 24 hr with DMSO and methotrexate used as negative and positive control, respectively. Thereafter, old medium was replaced with 200 μl MTT solution (final concentration of 0.5 mg/ml in PBS). Plates were incubated again for 4 hr after which the MTT solution was removed and formed formazan crystals were dissolved by adding 150 μl DMSO. Absorbance was measured at 570 nm and percentage of cell viability was calculated as [(absorbance of treated cells-absorbance of blank) / (absorbance of control cellsabsorbance of blank)] ×100).

CyQuant® direct cell proliferation assay
Dichloromethane fraction at 50 and 100 µg/ml was evaluated against MCF-7, A549 and PC3 using CyQuant® direct assay (Abraham et al., 2008). About 2000 cells were seeded in 48-well plates containing 400 μl of medium and incubated for 48 hr after which 0.2 μl of the fraction was added. Plates were incubated for another 24 hr and 200 μl of supernatant aspirated from each well was replaced with 200 μl of 2X detection reagent and plates were incubated again at 37°C for 60 min. Cells images were obtained using standard green filter microscope.

Caspase-3/7 green detection assay
Dichloromethane fraction (50 and 100 µg/ml) was evaluated using caspase-3/7 detection assay (Cobanoglu et al., 2016). About 2000 cells were seeded in 48-well plates containing 200 μl of medium and incubated for 48 hr after which 0.2 μl of the fraction was added. Plates were incubated at 37°C for 24 hr and the medium was replaced with 200 μl of caspase-3/7 green detection reagent. Plates were incubated again at 37°C for 30 min and imaging was done using standard microscope.

Statistical analysis
All experiments were done in triplicate and results are expressed as mean±standard errors of means. Statistical analysis was performed using GraphPad (Version 7.0, GraphPad Prism Software Inc., San Diego, CA). Sigmoid dose-response curve obtained from non-linear regression analyses (log of inhibitor versus normalized response) was used to determine the IC50. One-way ANOVA with Dunnett's multiple comparison tests was conducted at p<0.05 to detect difference in cytotoxicity of isolated compounds compared with methotrexate. Viability reducing ability of the isolated compounds was also compared with the negative control. Differences in the activity lower than p<0.05 were considered statistically significant.

Characterization of isolated compounds
Gradient CC and purification of the DCM fraction led to isolation of compounds 1-6 ( Figure 1).

CyQUANT® direct cell proliferation and caspase-3/7 green detection assays
In addition to MTT assay, the DCM fraction was evaluated by CyQuant® direct cell proliferation and caspase 3/7 green detection assays. The results are presented in Figures 2 and 3 respectively.

Discussion
The 1 H/ 13 C NMR spectra of C1, (methyl gallate) revealed an aromatic system exhibiting signals due to a pyrogallol ring at 6.94, 2H, (s) assigned to the chemically equivalent protons H-2/H-6 on carbons C-2/C-6 (δc 109.06). The methoxyl group at position C-7 was confirmed by HMBC correlation of a singlet OCH3 proton at 3.75 Hz (δc 52.15) to the carbonyl C-7 (δc 166.78). The 13 C NMR spectra revealed three phenolic groups attached to C-3, C-4, and C-5. The signal of chemically equivalent C-3 and C-5 appeared more downfield at (δ 146.0) compared with phenolic carbon at C-4 (δ 138.67) due to the combination of inductive and anisotropic effects of the ring on the carbon atom. This spectroscopic data is in agreement with literature (Sanchez et al., 2013).
The 13 C NMR spectra of C3 (132.9 (M + +H)), 3-amino-4-hydroxy-3methyldihydrofuran-2-one (benthamicone) indicated 5 carbon atoms. The most desheilded signal identified as carbonyl (δ 178.60) was assigned to C-2 and confirmed by DEPT spectra. 13 C NMR further revealed an oxygenated methylene at δc 62.91 assigned to C-5, an oxygenated methine carbon at δc 73.17 assigned to C-4, a quaternary carbon at δc 72.03 (C-3) attached to a primary amine and a methyl group at δc 21.74 assigned to C-6. The 1 H NMR spectra showed a methyl proton at δH 1.25 attached to C-3; an oxygenated methine proton at δH 4.01 attached to C-4; and an oxygenated methylene proton at δH 4.37 attached to C-5. In the HMBC, the position of the methyl group at 3a was confirmed by the correlation of the methyl protons at 1.25 ppm with carbon C-3 (δc 72.03); C-4 (δc 73.17); and C-5 (δc 62.91). These assignments were confirmed by COSY, HSQC, HMBC and DEPT.
The 13 C NMR spectra of C4 (2methoxyacrylic acid) indicated quaternary carbons at δc 168.27 and δc 145.18 assignable to C-1 and C-2, respectively; methoxy carbon with a chemical shift δc 50.10; and one sp2 methylene carbon at δc 108.86. The 1 H NMR spectrum revealed a singlet methoxy proton at δH 3.89 and a geminal proton signals at δH 5.52-5.50 (m) located on carbon C-3 (δc 108.86) as confirmed by DEPT and COSY. The carbonyl and hydroxyl groups were further confirmed by the IR spectra with signals at 1691 cm -1 and 3087 cm -1 , respectively.
In this study, three assays were used to assess fractions and compounds from C. benthamiana. The MTT assay showed DCM fraction as the most active on lung cancer cells followed by aqueous and hexane fractions (Table 7). Further investigation of DCM fraction by CyQUANT® direct assay showed reduction in the viability of cells and a progressive loss of cell membrane integrity (Figure 2). This dye is confirmed effective in evaluating anti-proliferative and cytotoxicity activity of drugs on cancer cells with the advantage of non-dependence on cell metabolic state (Quent et al., 2010). The caspase 3/7 assay also corroborated the observed activity of the DCM fraction. The fraction induced apoptosis in treated cells as shown by an increase in the number of dead cells. This was evident by a bright, fluorogenic green coloured dead cells in the treated groups compared to the untreated group (Payne et al., 2013). In some cases, a total rupture of the cell membrane was also observed (Figure 3).
The IC50 values of fractions and compounds indicated their inhibitory ability. These values were considered with the 95% confidence interval (CI) of the observed data (Tables 7 and 8). The CI is a function of the reliability of the IC50 generated from the data fitting to a doseresponse curve (Labelle et al., 2019). The calculated inhibitory concentration obtained from the dose-response curve clearly demonstrated the cytotoxic potentials of the fractions and the isolated compounds investigated compared with the standard drug.
Methyl gallate was previously reported as an anticancer, antiinflammatory and antioxidant agent (Sanchez et al., 2013). Its cytotoxic activity was also supported by Kamathama et al., (2015) with methyl gallate inhibiting A431 cells. The compound also reduced tumor growth in vitro and in tumor-bearing mice by decreasing CD4 + CD25 + Treg cell migration and reducing the suppressive function of effector T cells . Furthermore, caesalpinolida A and caesalpinolida B isolated from C. bonduc were reported to have cytotoxic activity against MCF-7 (Yadav et al., 2009). Also, brazilein isolated from C. sappan was reported active against HepG2 and Hep3B (liver), MDA-MB-231 and MCF-7 (breast), A549 (pulmonary), and CA9-22 (gingival) cells (Yen et al., 2010).
ANOVA with Dunnett's multiple test revealed that cytotoxicity of isolated compounds was not significantly different from that of methotrexate across the carcinoma cells. However, comparing the viability reducing ability of the isolated compounds with the negative control, the present study revealed that the isolated compounds elicited reduction in the number of viable cancer cells (Figure 5a-f), with varying potencies as reflected in their IC50 values (Tables 7 and 8).
Investigation of Caesalpinia benthamiana leaves led to the identification of six compounds including four previously unreported (2, 3, 5, and 6) and two known ones (1 and 4). The compounds had significant cytotoxic effect against MCF-7, A549 and PC3. Overall, the diterpenoid, had the highest cytotoxic activity across the cells. The use of C. benthamiana in traditional medicine for the treatment of cancer, was affirmed by this study.